Photon Factory Activity Report 2016 #34 (2017)

BL-5A, BL-17A/2015128

Crystal structures of highly simplified BPTIs provide insights into hydrationdriven increase of unfolding enthalpy
Mohammad Monirul Islama,b, Yutaka Kurodaa *aDepartment of Biotechnology and Life Sciences, Tokyo University of Agriculture and Technology,
Tokyo184-8588b
Department of Biochemistry and Molecular Biology, University of Chittagong, Chittagong-4331,
Bangladesh.

1 Introduction
Sequences encoding natural proteins constitute a tiny
fraction of the enormous variety of sequences that can bederived from the combination of 20 amino acids. Thisvariety is a major barrier to the elucidation of how aprotein structure is encoded in its sequence. However,artificial sequences encoding functional and stably folded
proteins have been designed from a reduced set of aminoacids or by specifying a reduced number of sites along theamino acid sequence. This suggests that the sequence
determinants for specifying a protein structure is limited,
and that the redundant information in a natural protein
sequence can be experimentally minimized withoutcompromising its native structure.
2 Experiments
All six BPTI variants were constructed using a pMMHa
vector by standard genetic engineering methods. X-ray
diffraction data were recorded from single crystals at thePhoton Factory, and structures were determined bymolecular replacement.
3 Results and Discussion
Here, we report a structural and thermodynamic analysis
of six extensively simplified BPTI variants, where 19–24
of its 58 residues are alanines, using differential scanningcalorimetry (DSC) and X-ray crystallography.
First, DSC indicated a two-state thermal unfolding,typical of a native protein with densely packed interior. Itwas surprising that a protein sequence where more than

40% of the residues are alanines can indeed fold into anative-like, well packed structure, which can be solved athigh resolution (Fig. 1). Next, comparison with thecrystal structures indicted that the enthalpy ofstabilization correlated with the increase of watermolecules recruited at places left by substituting largeside chains (Fig 2). These results provide new insights
into the hydration term of unfolding enthalpy and could
be rationalized by assuming that the water molecules
recruited nearby the alanines are stabilized both by thehydration shells of methyl groups and the formation of Hbonds between water molecules and protein atoms.
Overall, our results confirm and reinforce our previous
conclusion that the determinants of a protein are locatedinto the reduced sets of buried amino acids, whichsignificantly simplifies the protein folding problem.
Secondly, our report represents the first experimental
molecular “visualization” of a hydration-driven enthalpy
stabilization, which has been computationally predicted
but not demonstrated per se.

Fig 2: New hydration networks around the K15AR17A(left) site. Wt-BPTI and BPTI-24A are shown with aribbon model in blue and violet respectively. Spheres
represent water molecules around the alanine substitution
sites. (adapted from ref 1).

2.

numberof alanines
contained
in thesequences.
Fig 1: Structuresof thesimplified
BPTI
variants. This(a) wasin
sharp
contrasttoourinitialanticipationthat andtheSurface model of wt BPTI, BPTI-[5,55]st, BPTI-19A
replacement
oflongorbulkysidechainswiththesmall
BPTI-24A containing, respectively, 6, 8, 19 and 24
Surprisingly,
increasing
the Alanines
number ofarealanines
alanines out of58 residues.
shown induced
as blue
spheres (adapted from ref 1).

References
1) Crystal structures of highly simplified BPTIs provide
insights into hydration-driven increase of unfolding
enthalpy. Mohammad M Islam, Masafumi Yohda M,
Shun-Ichi Kidokoro, Yutaka Kuroda Scientific Reports 7,
Article number: 41205. (2017 Mar 7)2) The coordinates and structure factors of BPTI-21A,
BPTI-22Ab, BPTI-23A and BPTI-24A variants aredeposited in the Protein Data Bank under the PDB entrycodes 5JB4, 5JB5, 5JB6 and 5JB.

4.
5.

*ykuroda@cc.tuat.ac.jp